Soon after the advent of electrospray ionization mass spectrometry (ESI-MS) in the late 1980s, it became clear that this technique has an enormous potential for kinetic studies on solution-phase reactions.3, 4 Following the initiation of a (bio)-chemical process by mixing of two or more reactants, the kinetics can be monitored on-line, i.e., by direct injection of the reaction mixture into the ion source. The relative concentrations of multiple reactive species can be recorded as a function of time with extremely high sensitivity and selectivity. Transient intermediates may be identified based on their mass-to-charge ratio or their MS/MS characteristics. On-line ESI-MS kinetic studies have been carried out in a wide range of areas, including bioorganic chemistry,5, 6 enzymology,7-11 protein folding and assembly, 12, 13 and isotope exchange experiments in the context of protein conformational dynamics.14-19 
The use of ionization techniques other than ESI for on-line kinetic MS studies has been explored by a number of groups.20-22 Due to its versatility, however, ESI-MS remains by far the most popular technique for studies of this kind. An alternative approach for kinetic measurements involves the use of quench-flow techniques in conjunction with off-line MS analysis.23, 24 In quench-flow experiments, the reaction is initiated by rapid mixing of the reactants, followed by mixing with a quenching agent, such as acid, base, or organic solvent, that abruptly stops the reaction after a specified period of time. An advantage of that technique is the possible incorporation of purification steps in situations where components of the reaction mixture would interfere with the MS analysis. Quench-flow methods undoubtedly represent a powerful tool for kinetic studies, but they can be problematic in cases where reactive species are not stable under the conditions of the quenched reaction mixture. Also, quench-flow studies are laborious because individual time points have to be measured in separate experiments.
Of particular importance for studies on a wide range of chemical and biochemical systems are techniques capable of providing kinetic data on rapid time scales, i.e., seconds to milliseconds or even microseconds.25 On-line ESI-MS methods have been used for characterizing processes with half-lives down to roughly 30 ms.16 This temporal resolution is orders of magnitude lower than that obtainable in rapid-mixing experiments with optical detection.26, 27 It therefore appears that there might still be considerable room for extending the time range that is accessible to MS-based kinetic techniques.
On-line kinetic studies can be carried out in two different modes of operation: (i) In “kinetic mode”, the abundance of one or more species is monitored as a function of time, e.g., by monitoring the intensity at selected m/z values on a quadrupole mass analyzer. This type of experiment provides detailed intensity-time profiles for individual reactive species, which allows the accurate determination of rate constants. Stopped-flow ESI-MS is a method capable of providing highly accurate data in kinetic mode.28, 29 Unfortunately, this approach requires prior knowledge of the m/z value(s) of interest, thus posing a serious limitation for studies on processes that involve unknown intermediates. Also the stopped-flow ESI-MS has inherent time resolution limitations and hence so far it has not been possible to extend this technique below the range of ˜O./S. (ii) For experiments carried out in “spectral mode”, entire mass spectra are recorded for selected reaction times, which allows the detection and identification of transient intermediates. The use of stopped-flow ESI-MS for studies in spectral mode is difficult, because entire mass spectra would have to be recorded on a millisecond time scale, which poses a challenge even for time-of-flight instruments or quadrupole ion traps. Experiments in spectral mode are more easily carried out by using continuous-flow methods. In contrast to stopped-flow ESI-MS, this approach does not involve real-time data acquisition; spectral mode data can therefore be recorded even with slow-scanning mass analyzers.5, 12, 15, 30, 31 Usually, the reaction chamber in continuous-flow studies is a capillary that is mounted between a mixer and the ESI source. The reaction time is determined by the capillary dimensions and by the solution flow rate. Controlling the reaction time by changing the solution flow rate is not advisable because this may result in artifactual changes of analyte ion abundances. Reaction capillaries of different; length are therefore most commonly used for recording spectra at different times points. A drawback of existing continuous-flow methods is the difficulty of obtaining intensity-time profiles of selected ions. These kinetic mode data have to be “pieced together” from multiple measurements carried out with different capillary lengths, in a manner analogous to quench-flow studies.
Thus, it was desired to improve capillaries for mixing reactant solutions for ESI-MS based reaction analyses.